The present invention is directed to novel inhibitory compounds which are capable of reducing, eliminating or preventing human immunodeficiency virus (HIV) infection and which are isolated from CD8+ lymphocytes. The invention is also directed to CD8+ cell lines which secrete these novel inhibitors. The invention is further directed to compositions comprising an inhibitor of the invention and to methods for the use of such compositions in the prevention and/or treatment of HIV infection.
CD8+ cells from HIV-1 infected individuals exhibit HIV-1 inhibitory activity that is not MHC-restricted and is non-cytolytic (Walker et al., Science 234:1563-1566, 1986; Walker et al., J. Virol. 65:5921-5927, 1991; Mackewicz et al., Cell. Immunol. 153:329-343, 1993). The inhibition does not require cell to cell contact and is mediated through soluble factors, hence the original name CD8+-cell antiviral factors (CAF, Walker et al., J. Virol. 65:5921-5927, 1991). Further studies suggested that CD8+ CAF activity correlated with the clinical status of an infected individual (Blackbourn et al., Proc. Natl. Acad. Sci. USA 93:13125-13130, 1996; Mackewicz et al., J. Clin. Invest. 87:1462-1466, 1991). The mechanism of action of this activity, as originally described, was thought, at least partially, to be through the inhibition of Tat-induced transcription (Mackewicz et al., Proc. Natl. Acad. Sci. USA 92:2308-2312, 1995; Chen et al., AIDS Res. Human Retroviruses 9(11):1079-1086, 1993). Some insight into this soluble HIV-1 inhibitory activity has recently been gained by the observation that a cocktail of CC chemokines, RANTES, MIP-1xcex1 and MIP-1xcex2 (released by HTLV-I transformed CD8+ cells) effectively block replication of HIV in CD4+ lymphocytes (Cocchi et al., Science 270:1811-1815, 1995). These chemokines are naturally made by a number of immune cells including CD8+ cells. This inhibitory activity was observed against macrophage (M) tropic strains of virus but not against T-lymphocyte (T) tropic laboratory adapted strains of HIV (i.e., the laboratory adapted strain HIV IIIB). The simultaneous discovery of the HIV-1 inhibitory effects of chemokines and the isolation of fusin (or CXCR4) as a co-receptor for T-tropic strains of HIV-1 (Feng et al., Science 272:872-877, 1996) led to the discovery of a series of chemokine receptors that can act as co-receptors for entry of certain strains of HIV-1 into susceptible cells (Deng et al., Nature 381:661-666, 1996; Dragic et al., Nature 381:667-673,; Doranz et al., Cell 85:1149-1158, 1996; Marcon et al., J. Virol. 71:2522-2527, 1997; Chen et al., J. Virol. 71:2705-2714, 1997).
The mechanism of HIV-1 inhibition by the cocktail, RANTES, MIP-1xcex1 and MIP-1xcex2 is via the recently described second HIV-1 co-receptor, CCR-5, which is a ligand for each of these inhibitory chemokines. CCR-5 is a receptor for entry of M-tropic isolates into T-cells but not for entry of T-cell tropic strains (Deng et al., Nature 381:661-666, 1996; Dragic et al., Nature 381:667-673, 1996). The latter strains utilize the CXC chemokine receptor, CXCR4 or fusin and infection can be blocked with the ligand for CXCR4, SDF-1 (Oberlin et al., Nature 382:833-835, 1996). Additional members of the chemokine receptor family can be utilized by strains of HIV-1 and the related viruses HIV-2 and SIV to gain entry into cells (Doranz et al., Cell 85:1149-1158, 1996; Marcon et al., J. Virol. 71:2522-2527, 1997; Chen et al., J. Virol. 71:2705-2714, 1997; Liao et al., J.Exp.Med. 185:2015-2023, 1997). Two new members of the chemokine seven-transmembrane G-protein-coupled receptor family have recently been identified by expression cloning utilizing the SIV envelope protein. These receptors (Bob and Bonzo), expressed in lymphoid tissue, can be used by SIV as well as strains of HIV-2 and M-tropic HIV-1. The natural ligands for these receptors have yet to be identified (Deng et al., Nature 388:296-300, 1997). Early work on soluble CD8+ factors suggested that the CC class of chemokines may not be the only potent inhibitors released from CD8+ cells. The inhibitory activity found in CD8+ supernatants appeared to be against a broader range of HIV-1 isolates including both M-tropic and T-tropic viruses and appeared to at least in part inhibit HIV-1 transcription (Mackewicz et al., Proc. Natl. Acad. Sci. USA 92:2308-2312, 1995; Chen et al., AIDS Res. Human Retroviruses 9(11):1079-1086, 1993; Moriuchi et al., Proc. Natl. Acad. Sci. USA 93:15341-15345, 1996; Paliard et al., AIDS 10:1317-1321, 1996). In contrast, the chemokine cocktail of RANTES, MIP-1xcex1 and MIP-1xcex2 is believed to work primarily at the point of viral entry in the cell and is effective only against M-tropic strains of HIV-1. Additional inhibitory factors recently described include the HHV-8 chemokine homologue, vMipII, and macrophage-derived chemokine (MDC). vMipII acts like a receptor antagonist with binding to both CC as well as CXC chemokine receptors including CCR3, CCR5 and CXCR4 and, as predicted, has broad antiviral activity (Kiedal et al., Science 277:1656-1659,1997). MDC was isolated from HTLV-I immortalized CD8+ cell lines. Predominantly made in activated PBMC (macrophages as well as CD8+ cells), this factor has broad activity against T-tropic and M-tropic strains. While MDC inhibits entry into macrophages and T-cells, it has no effect against the same strains in the transformed T-cell line, PM-1. Native MDC has inhibitory activity in the nanogram (ng) to microgram (xcexcg) range (Pal et al., Science 278:695-698, 1997). Studies have demonstrated that CD8+-derived HIV-1 inhibitory activity from either primary or transformed cells is not blocked by neutralizing antibodies against xcex2-chemokines and is not related to differences in MIP-1xcex1, MIP-1xcex2 and RANTES levels (Moriuchi et al., Proc. Natl. Acad. Sci. USA 93:15341-15345, 1996; Paliard et al., AIDS 10:1317-1321, 1996). Therefore, CD8+ cells appear to make a number of natural HIV-1 inhibitory factors and further characterization of these factors should not only provide leads into new classes of inhibitory drugs against HIV-1 but will provide further understanding of host factors that might be play a role in controlling replication in certain individuals. This is best exemplified by the discovery of the inhibitory chemokines described above and the co-receptor CCR5. It has subsequently been determined that T-cells of certain individuals containing deletion mutations (xcex9432) in both alleles of the CCR5 receptor are highly resistant to infection in vitro with M-tropic strains of HIV-1 (Lin et al., Cell 86:367-377, 1996). In large population studies of HIV-1 infected individuals, carriers of this double deletion are extremely rare while the rates in other populations are 1% (Dean et al., Science 273:1856-1862, 1996). Individuals who are heterozygous for this deletion appear to become infected but have a somewhat slower progression to disease (Huang et al., Nature Med. 2:1240-1243, 1996). Furthermore, mutations in another co-receptor, CCR2b, while affording no protection from infection, confer some protection from disease progression (Smith et al., Science 277:959-965, 1997). Thus, further characterization of natural inhibitors has clearly impacted the field in both the area of understanding host factors that play a role in infection and progression as well as leading the way to a whole new class of inhibitors based on the chemokine/co-receptor interaction. The present invention is based on the discovery of novel natural inhibitors of HIV-1 replication isolated from CD8+ cells.
The present invention is directed to novel inhibitors of HIV replication which are capable of reducing, eliminating or preventing HIV infection and which are isolated from CD8+ lymphocytes. The invention is also directed to CD8+ cell lines which secrete these novel inhibitors. The invention is further directed to compositions comprising an inhibitor of the invention and to methods for the use of such compositions in the prevention and/or treatment of HIV infection.